Label-free functional nucleic acid sensors for detecting target agents

Abstract

A general methodology to design label-free fluorescent functional nucleic acid sensors using a vacant site approach and an abasic site approach is described. In one example, a method for designing label-free fluorescent functional nucleic acid sensors (e.g., those that include a DNAzyme, aptamer or aptazyme) that have a tunable dynamic range through the introduction of an abasic site (e.g., dSpacer) or a vacant site into the functional nucleic acids. Also provided is a general method for designing label-free fluorescent aptamer sensors based on the regulation of malachite green (MG) fluorescence. A general method for designing label-free fluorescent catalytic and molecular beacons (CAMBs) is also provided. The methods demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes. Sensors and methods of using the disclosed sensors are also provided.

We claim: 
     
       1. A sensor, comprising
 a catalytic nucleic acid molecule specific for a target agent comprising an enzyme nucleic acid strand and a substrate nucleic acid strand, 
 wherein the enzyme nucleic acid strand comprises a 3′-end and a 5′-end and an active site specific for a target agent, 
 wherein the substrate nucleic acid strand comprises a 3′-end and a 5′-end, 
 wherein the substrate nucleic acid strand comprises nucleotides at the 5′-end of the substrate nucleic acid strand that permits formation of a loop of at least six nucleotides at the 5′-end of the substrate nucleic acid strand, and 
 wherein the substrate nucleic acid strand hybridizes with the enzyme nucleic acid strand, thereby forming a vacant site between the 3′-end of the enzyme nucleic acid strand and the 5′-end of the substrate nucleic acid strand, wherein the vacant site is opposite to a cytosine present in the substrate nucleic acid strand and wherein the vacant site can bind to a fluorophore, 
 wherein in the absence of the target agent, fluorescence of the fluorophore when bound to the vacant site is quenched, and wherein in the presence of the target agent, catalytic cleavage of substrate nucleic acid strand perturbs the vacant site and releases the fluorophore bound to the vacant site resulting in increased fluorescence. 
 
     
     
       2. The sensor of  claim 1 , wherein the vacant site is flanked by guanines present on a 3′-nucleotide of the enzyme nucleic acid strand and a 5′-nucleotide of the substrate nucleic acid strand. 
     
     
       3. The sensor of  claim 1 , wherein at least one pair of nucleotides are mismatched upon hybridization of the substrate nucleic acid molecule with the enzyme nucleic acid strand. 
     
     
       4. The sensor of  claim 1 , wherein the catalytic nucleic acid comprises a DNAzyme or an aptazyme. 
     
     
       5. The sensor of  claim 1 , wherein the sensor is attached to a solid support. 
     
     
       6. A kit comprising:
 the sensor of  claim 1 ; and 
 one or more of a buffer, a chart for correlating detected fluorescence and amount of target agent present, or a test agent. 
 
     
     
       7. A method for detecting a target agent, comprising
 contacting the sensor of  claim 1  with a sample under conditions sufficient to allow the target agent in the sample to bind to the sensor resulting in cleavage of the sensor or a conformational change of the sensor; and 
 detecting fluorescence, wherein detection of fluorescence indicates the presence of the target agent in the sample, and an absence of detected fluorescence indicates the absence of the target agent in the sample. 
 
     
     
       8. The method of  claim 7 , further comprising quantifying the target agent, wherein a level of fluorescence detected indicates an amount of target agent present. 
     
     
       9. The sensor of  claim 1 , wherein the catalytic nucleic acid comprises a DNAzyme. 
     
     
       10. The sensor of  claim 1 , wherein the catalytic nucleic acid comprises an RNAzyme. 
     
     
       11. The sensor of  claim 1 , wherein the loop is formed by at least 15 nucleotides between the vacant site and the cytosine opposite to the vacant site. 
     
     
       12. The sensor of  claim 1 , wherein the target agent is a metal. 
     
     
       13. The sensor of  claim 1 , wherein the target agent is a heavy metal. 
     
     
       14. The sensor of  claim 1 , wherein the target agent is mercury, cadmium, arsenic, chromium, thallium, magnesium, copper, lead or uranium. 
     
     
       15. The sensor of  claim 1 , wherein the target agent is lead or uranium. 
     
     
       16. The sensor of  claim 1 , wherein the target agent is a pathogen, protein, recreational drug, or cell. 
     
     
       17. The sensor of  claim 1 , wherein the fluorophore is quenched in the presence of cytosine. 
     
     
       18. The sensor of  claim 1 , wherein the fluorophore is 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND). 
     
     
       19. The sensor of  claim 1 , wherein the fluorophore is 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC), fluorescein, rhodamine, malachite green (MG) or an Alexa Fluor fluorophore.